Current techniques for biomolecule transfer are a primary bottleneck in intracellular signal manipulation in laboratory research, drug delivery and gene therapy. Typically, exogenous nucleic acids (e.g., plasmid DNA, antisense RNA, siRNA, etc.) can be used to alter intracellular gene expression upon delivery across biological membranes. Such a process, commonly referred to as transfection, can be carried out with a variety of techniques including calcium phosphate transfection, lipofection, microinjection, micropipette dialysis, microparticle bombardment, electroporation, virus infection, etc. Generally, these technologies are categorized as viral or non-viral delivery techniques.
Viral delivery techniques have generally proven to be superior to non-viral delivery techniques in terms of efficiency. However, the viral delivery techniques are intrinsically bio-hazardous, non-specific, and can trigger side effects in the host. Therefore, viral delivery techniques for cell transfection are generally less desirable than non-viral delivery techniques. This is particularly true for gene therapy and other in vivo applications.
Non-viral delivery techniques are generally less bio-hazardous and tend to trigger less undesirable side effects in a host. However, non-viral delivery techniques are typically less efficient than viral delivery techniques. This inefficiency in non-viral delivery techniques is typically the result of low cell viability after non-viral transfection. Such low cell viability is typically the result of cell damage resulting from mechanical impact, electric shock and/or the toxicity of the chemicals used during non-viral transfection. In other words, trauma to the cell during non-viral transfection typically results in low cell viability and, therefore, low efficiency in non-viral techniques.
Another problem associated with altering intracellular gene expression occurs when using plasmid DNAs (and/or other exogenous nucleic acids). In order to alter the expression of the target gene, the plasmids (or other exogenous nucleic acids) must ultimately enter the nucleus of the cell. However, in order to enter the nucleus, the plasmids (or other exogenous nucleic acids) have to overcome three fundamental intracellular defenses. The first defense is the cell membrane, which must be penetrated. The second defense is the lysosomes located in the cellular fluid, e.g., cytosol. Once the plasmids (or other exogenous nucleic acids) have penetrated the cell membrane, they must travel through the cellular fluid, where the lysosomes may hydrolyze the plasmids (or other exogenous nucleic acids). The third defense is the nucleus envelope, which must also be penetrated. Most transfection techniques merely generate passageways for molecules to penetrate the outer cell membrane, and are not capable of helping the molecules overcome the other intracellular defenses.
Still another problem associated with altering intracellular gene expression is the variability in the effectiveness of the transfection techniques in various cell types. More particularly, there are dramatic variances in the transfection efficiency among different cell types. Certain kinds of cells, particularly the non-dividing cells, are generally regarded as “difficult-to-transfect” cells. Significantly, these types of cells are generally those with the greatest biological significance, and hence of greatest interest with respect to altering intracellular gene expression.
To overcome the foregoing limitations of the various transfection techniques, nanomaterials have been utilized to facilitate penetration of biological membranes. Carbon nanotubes, for example, have been engulfed by the cells through the endocytotic process or by an unidentified mechanism.
Additionally, an array of carbon nanotubes, grown on a substrate, has been used as a “nail board”. The target cells were positioned against the nanotube tips and then a mechanical force was applied, causing the cells to be impaled on the tubes. During this procedure, despite the trauma of impalement, the cells remained viable.
In addition, metal nanorods have been shown to penetrate cells by receptor-mediated endocytosis. One technique, sometimes referred to as magnetofection, utilizes magnetic nanoparticles to help concentrate the particle-DNA-liposome composites adjacent to the cell surface, and subsequently improves biomolecule trasfection.
Another technique, sometimes referred to as calcium phosphate transfection, takes advantage of cellular biomineralization to introduce DNA into the cells. More particularly, it has been shown that well-defined calcium phosphate-DNA nanostructures have increased transfection efficacy.
In these latest developments in nano-biotechnology, nanostructures have demonstrated promising characteristics for biomolecule delivery. A significant advantage of nanostructures is their nanoscale dimension, which facilitates high cell viability during the transfection process. In particular, the small size of the nanostructures allows them to utilize normal biological processes (e.g., endocytosis, biomineralization, etc.) to provide entry into the cells. This characteristic makes nanostructures good candidates to carry molecules across cell membranes.
It should be appreciated that, in order to carry molecules, the surfaces of the nanostructures must be adapted so that molecules can be bound onto the nanostructures by covalent bonding, electrostatic attraction, hydrophobic adsorption, etc.
Thus, to date, a number of new techniques have been developed to utilize nano-biotechnology for molecule delivery. However, all of these techniques suffer from one or more significant disadvantages, e.g., slow transfection speed, significant cell damage during transfection, difficulties binding the molecules to the nanomaterials, etc.,
In addition to the foregoing, because the mechanisms involved in introducing nanostructures into cells through biopassages are not completely understood, it is desirable to have a more controlled and directed way of passing nanostructures into cells. Additionally, because intracellular gene expression requires biological molecules to penetrate the nucleus of the cell, there exists a need for a more reliable method for passing the biological molecules through the cell membrane and ultimately into the nucleus.